A survey of electrokinetically-driven microfluidics for cancer cells manipulation

Cancer is one of the leading causes of annual deaths worldwide, accounting for nearly 10 million deaths each year. Metastasis, the process by which cancer spreads across the patient's body, is the main cause of death in cancer patients. Because the rising trend observed in statistics of new cancer cases and cancer-related deaths does not allow for an optimistic viewpoint on the future—in relation to this terrible disease—the scientific community has sought methods to enable early detection of cancer and prevent the apparition of metastatic tumors. One such method is known as liquid biopsy, wherein a sample is taken from a bodily fluid and analyzed for the presence of CTCs or other cancer biomarkers (e.g., growth factors). With this objective, interest is growing by year in electrokinetically-driven microfluidics applied for the concentration, capture, filtration, transportation, and characterization of CTCs. Electrokinetic techniques—electrophoresis, dielectrophoresis, electrorotation, and electrothermal and EOF—have great potential for miniaturization and integration with electronic instrumentation for the development of point-of-care devices, which can become a tool for early cancer diagnostics and for the design of personalized therapeutics. In this contribution, we review the state of the art of electrokinetically-driven microfluidics for cancer cells manipulation.     

Analysis of microorganisms with nonlinear electrokinetic microsystems

Nonlinear electrokinetics (EK), specifically electrophoresis of the second kind, dielectrophoresis (DEP) and electrorotation (EROT), have gained significant interest recently for their flexibility and labeless discriminant manner of operation. The current applications of these technologies are a clear advancement from what they were when first discovered, but also still show strong signs of future growth. The present review article presents a discussion of the current uses of microscale nonlinear EK technologies as analytical, sensing, and purification tools for microorganisms. The discussion is focused on some of the latest discoveries with various nonlinear EK microfluidic techniques, such as DEP particle trapping and EROT for particle assessments, for the analysis of microorganisms ranging from viruses to parasites. Along the way, special focus was given to key research articles from within the past two years to provide the most up-to-date knowledge on the current state-of-the-art within the field of microscale EK, and from there, an outlook on where the future of the field is headed is also included.     

Particle trapping in electrically driven insulator-based microfluidics: Dielectrophoresis and induced-charge electrokinetics

Electrokinetically driven insulator-based microfluidic devices represent an attractive option to manipulate particle suspensions. These devices can filtrate, concentrate, separate, or characterize micro and nanoparticles of interest. Two decades ago, inspired by electrode-based dielectrophoresis, the concept of insulator-based dielectrophoresis (iDEP) was born. In these microfluidic devices, insulating structures (i.e., posts, membranes, obstacles, or constrictions) built within the channel are used to deform the spatial distribution of an externally generated electric field. As a result, particles suspended in solution experience dielectrophoresis (DEP). Since then, it has been assumed that DEP is responsible for particle trapping in these devices, regardless of the type of voltage being applied to generate the electric field—direct current (DC) or alternating current. Recent findings challenge this assumption by demonstrating particle trapping and even particle flow reversal in devices that prevent DEP from occurring (i.e., unobstructed long straight channels stimulated with a DC voltage and featuring a uniform electric field). The theory introduced to explain those unexpected observations was then applied to conventional “DC-iDEP” devices, demonstrating better prediction accuracy than that achieved with the conventional DEP-centered theory. This contribution summarizes contributions made during the last two decades, comparing both theories to explain particle trapping and highlighting challenges to address in the near future.     

Dielectrophoretic manipulation of particle mixtures employing asymmetric insulating posts

A novel scheme for particle separation with insulator‐based dielectrophoresis (iDEP) was developed. This technique offers the capability for an inverted order in particle elution, where larger particles leave the system before smaller particles. Asymmetrically shaped insulating posts, coupled with direct current (DC) biased low‐frequency alternating current (AC) electric potentials, were used to successfully separate a mixture of 500 nm and 1 μm polystyrene particles (size difference of 0.5 μm in diameter). In this separation, the 1 μm particles were eluted first, demonstrating the discriminatory potential of this methodology. To extend this technique to biological samples, a mixture containing Saccharomyces cerevisiae cells (6.3 μm) and 2 μm polystyrene particles was also separated, with the cells being eluted first. The asymmetric posts featured a shorter sharp half and a longer blunt half; this produced an asymmetry in the forces exerted on the particles. The negative DC offset produced a net displacement of the smaller particles toward the upstream direction, while the post asymmetry produced a net displacement of the larger particles toward the downstream direction. This new iDEP approach provides a setup where larger particles are quickly concentrated at the outlet of the post array and can be released first when in a mixture with smaller particles. This new scheme offers an extra set of parameters (alternating current amplitude, DC offset, post asymmetry, and shape) that can be manipulated to obtain a desired separation. This asymmetric post iDEP technique has potential for separations where it is important to quickly elute and enrich larger and more fragile cells in biological samples.     

On the recent developments of insulator‐based dielectrophoresis: A review

Insulator‐based dielectrophoresis (iDEP), also known as electrodeless DEP, has become a well‐known dielectrophoretic technique, no longer viewed as a new methodology. Significant advances on iDEP have been reported during the last 15 years. This review article aims to summarize some of the most important findings on iDEP organized by the type of dielectrophoretic mode: streaming and trapping iDEP. The former is primarily used for particle sorting, while the latter has great capability for particle enrichment. The characteristics of a wide array of devices are discussed for each type of dielectrophoretic mode in order to present an overview of the distinct designs and applications developed with iDEP. A short section on Joule heating effects and electrothermal flow is also included to highlight some of the challenges in the utilization of iDEP systems. The significant progress on iDEP illustrates its potential for a large number of applications, ranging from bioanalysis to clinical and biomedical assessments. The present article discusses the work on iDEP by numerous research groups around the world, with the aim of proving the reader with an overview of the state‐of‐the‐art in iDEP microfluidic systems.     

Polarization behavior of polystyrene particles under direct current and low‐frequency (<1 kHz) electric fields in dielectrophoretic systems

The relative polarization behavior of micron and submicron polystyrene particles was investigated under direct current and very low frequency (<1 kHz) alternating current electric fields. Relative polarization of particles with respect to the suspending medium is expressed in terms of the Clausius–Mossotti factor, a parameter of crucial importance in dielectrophoretic‐based operations. Particle relative polarization was studied by employing insulator‐based dielectrophoretic (iDEP) devices. The effects of particle size, medium conductivity, and frequency (10–1000 Hz) of the applied electric potential on particle response were assessed through experiments and mathematical modeling with COMSOL Multiphysics^®. Particles of different sizes (100–1000 nm diameters) were introduced into iDEP devices fabricated from polydimethylsiloxane (PDMS) and their dielectrophoretic responses under direct and alternating current electric fields were recorded and analyzed in the form of images and videos. The results illustrated that particle polarizability and dielectrophoretic response depend greatly on particle size and the frequency of the electric field. Small particles tend to exhibit positive DEP at higher frequencies (200–1000 Hz), while large particles exhibit negative DEP at lower frequencies (20–200 Hz). These differences in relative polarization can be used for the design of iDEP‐based separations and analysis of particle mixtures.     

Reorientation of microfluidic channel enables versatile dielectrophoretic platforms for cell manipulations

Dielectrophoresis is a versatile tool for the sorting, immobilization, and characterization of cells in microfluidic systems. The performance of dielectrophoretic systems strongly relies on the configuration of microelectrodes, which produce a nonuniform electric field. However, once fabricated, the microelectrodes cannot be reconfigured to change the characteristics of the system. Here, we show that the reorientation of the microfluidic channel with respect to the microelectrodes can be readily utilized to alter the characteristics of the system. This enables us to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, change the deflection pattern of nonviable cells along the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. We demonstrate that the reorientation of the microfluidic channel is an effective tool to create versatile dielectrophoretic platforms using the same microelectrode design.     

A high-throughput dielectrophoresis-based cell electrofusion microfluidic device

A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO? film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (～12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.     

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Microfluidic device embedding electrodes for dielectrophoretic manipulation of cells‐A review

Microfluidic device embedding electrodes realizes cell manipulation with the help of dielectrophoresis. Cell manipulation is an important technology for cell sorting and cell population purification. Till now, the theory of dielectrophoresis has been greatly developed. Microfluidic devices with various arrangements of electrodes have been reported from the beginning of the single non‐uniform electric field to the later multiple physical fields. This paper reviews the research status of microfluidic device embedding electrodes for cell manipulation based on dielectrophoresis. Firstly, the working principle of dielectrophoresis is explained. Next, cell manipulation approaches based on dielectrophoresis are introduced. Then, different types of electrode arrangements in the microfluidic device for cell manipulation are discussed, including planar, multilayered and microarray dot electrodes. Finally, the future development trend of the dielectrophoresis with the help of microfluidic devices is prospected. With the rapid development of microfluidic technology, in the near future, high precision, high throughput, high efficiency, multifunctional, portable, economical and practical microfluidic dielectrophoresis will be widely used in the fields of biology, medicine, agriculture and so on.     

Effects of microchannel geometry on preconcentration intensity in microfluidic chips with straight or convergent-divergent microchannels

Preconcentration microfluidic devices are fabricated incorporating straight or convergent-divergent microchannels and hydrogel or Nafion membranes. Sample preconcentration is achieved utilizing concentration-polarization effects. The effects of the microchannel geometry on the preconcentration intensity are systematically examined. It is shown that for the preconcentrator with the straight microchannel, the time required to achieve a satisfactory preconcentration intensity increases with an increasing channel depth. For the convergent-divergent microchannel, the preconcentration intensity increases with a reducing convergent channel width. Comparing the preconcentration performance of the two different microchannel configurations, it is found that for an equivalent width of the main microchannel, the concentration effect in the convergent-divergent microchannel is faster than that in the straight microchannel.     

Fast immobilization of probe beads by dielectrophoresis-controlled adhesion in a versatile microfluidic platform for affinity assay

The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s--depending on the protocol--to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.     

Electroporation of cells in microfluidic devices: a review

In recent years, several publications on microfluidic devices have focused on the process of electroporation, which results in the poration of the biological cell membrane. The devices involved are designed for cell analysis, transfection or pasteurization. The high electric field strengths needed are induced by placing the electrodes in close proximity or by creating a constriction between the electrodes, which focuses the electric field. Detection is usually achieved through fluorescent labeling or by measuring impedance. So far, most of these devices have only concerned themselves solely with the electroporation process, but integration with separation and detection processes is expected in the near future. In particular, single-cell content analysis is expected to add further value to the concept of the microfluidic chip. Furthermore, if advanced pulse schemes are employed, such microdevices can also enhance research into intracellular electroporation.     

Capillary-based microfluidic analysis systems

An overview of the recent development of high-throughput capillary-based microfluidic analysis systems (CBMAS) is presented. The advantages and applications of these systems are discussed and illustrated by recent results obtained in the authors’ group and other groups. A possible outlook for CBMAS is discussed.     

Recent advances in dielectrophoresis-based cell viability assessment

Dielectrophoresis (DEP) is a non-destructive, accurate, and label-free cell manipulating technique and DEP applications have been found in various fields. Assessment of cell viability is one of the important applications and many investigations have been reported. In this paper, cell polarization and its modeling, some key parameters employed for living/dead cell separation, as well as electrode configurations are reviewed. Focus is given to the latest development of DEP devices employed for the assessment of cell viability. Experimentally determined factors for separating living/dead cells, such as the conductivity of suspending medium and the frequency of applied electric field, are summarized. The future directions and potential challenges in this field are also outlined.     

Nanoparticle-based assays in automated flow systems: A review

Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities.     

Actuation of adaptive liquid microlens droplet in microfluidic devices: A review

A significant growth of research on adaptive liquid lens is achieved over the past decades, and the field is still attracting increasing attentions, focusing on the transition from the current stage to the commercialized stage. The challenges faced are not limited to fabrication, material, small tuning range in focal lengths, additional control systems, limitations in special actuation methods and so on. In addition, the use of external driving parts or systems induce extra problem on bulky appearance, high cost, low reliability etc. Therefore, adaptive liquid lens will be an interesting research focus in both microfluidics and optofluidics science. This review attempts to summarize and focus on the droplet profile deformation under different driving mechanisms in tunable liquid microlens as well as the application in cameras, cell phone and so on. The driving techniques are generally categorized in terms of mechanisms and driving sources.     

Analytical treatment of electrokinetic microfluidics in hydrophobic microchannels

Microfluidics in microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS) devices is complex due to the large surface area to volume ratio. Thus, surface properties play an important role in flow behavior. In this paper, we summarize the effects of electric double layer and surface hydrophobicity of rectangular microchannels on time-dependent electrokinetic flow. Theoretically, we have shown that flow resistance can, in principal, be significantly reduced so that a larger flow rate can be obtained for pressure-driven flow or electric-field-driven flow. This relies on the ability to change surface charges and surface hydrophobicity independently. Our theoretical results provide guidelines for the design and operation of microfluidic flow in rectangular microchannels. Because of liquid slippage, zeta potential determination by traditional method could be overestimated. Taking into account the effect of hydrophobicity, a modified method is proposed to determine the zeta potential and slip coefficient for parallel-plate microchannels with hydrophobic surfaces.     

Polymer microfabrication technologies for microfluidic systems

Polymers have assumed the leading role as substrate materials for microfluidic devices in recent years. They offer a broad range of material parameters as well as material and surface chemical properties which enable microscopic design features that cannot be realised by any other class of materials. A similar range of fabrication technologies exist to generate microfluidic devices from these materials. This review will introduce the currently relevant microfabrication technologies such as replication methods like hot embossing, injection molding, microthermoforming and casting as well as photodefining methods like lithography and laser ablation for microfluidic systems and discuss academic and industrial considerations for their use. A section on back-end processing completes the overview.